Technical Abstract Sex Steroids and Runx Signaling in Bone The following contains proprietary/privileged information that Dr. Frenkel requests not to be released to persons outside the Government, except for purposes of review and evaluation. Runx2 is a master osteoblast transcription factor playing pivotal roles in skeletal development and homeostasis. In humans, Runx2 haplotypes contribute to variations in bone mass. Runx1, which is expressed in osteoblasts and shares similar DNA-binding properties with Runx2, has been implicated in bone metabolism as well. Sex steroid hormones and their receptors (SHRs) also play critical roles in bone health and disease, and are targets for existing and developing drugs that affect bone mass and fragility either positively or negatively. The proskeletal effects of sex steroids are mediated by anabolic effects in osteoblasts, but more importantly by attenuating bone resorption. The anti-resorptive effects of sex steroids are attributable to both direct pro-apoptotic action in osteoclasts and indirect inhibition of bone turnover via poorly understood mechanisms in osteoblasts and other mesenchymal cells. We found that the activated estrogen receptor [unreadable] (ER[unreadable]) and the androgen receptor (AR) each inhibits Runx2, and that AR, but not ER[unreadable], inhibits Runx1 as well. These inhibitory activities are important in light of recent data from [unreadable]1(I)collagen-Runx2 transgenic mice, indicating that Runx2 must be restrained in order to keep bone turnover in check and prevent osteoporosis. We therefore propose to investigate in depth the physical interactions between Runx proteins and SHRs, the mechanisms mediating the resulting inhibition of Runx2 and/or Runx1, and the in vivo physiological implications. This will be done by analyses of recombinant and transiently expressed proteins, as well as the endogenous SHR and Runx proteins in osteoblasts, including their associations with each other, with co- regulators, and with genomic Runx targets. Specific Aim 1 is to dissect the functional and molecular interactions between ER[unreadable] and Runx2. Specific Aim 2 is to dissect the functional and molecular interactions between AR and Runx2, as well as between AR and Runx1. Based on our preliminary data, we hypothesize the existence of both similar and unique features for each of these interactions. Specific Aim 3 is to test the ability of estrogens (and later androgens) to correct the hyper-osteoclastogenic phenotype of osteoblasts over- expressing Runx2 in vivo and when co-cultured with osteoclasts. Incorporated into Aims 1-3 are experiments addressing novel mechanisms of action of selective estrogen receptor modulators (SERMs). Like estradiol, SERMs promote a physical interaction between ER[unreadable] and Runx2. However, SERMs elicit different functional outcomes, possibly explaining the variable skeletal effects of these drugs. Our studies will provide novel insights into the regulation of skeletal metabolism by sex hormones, and will reveal commonalities and differences between the genders at the molecular level. They will decipher cryptic mechanisms of action of existing SERMs, and support the rationale development of novel ones, based on their influence on Runx proteins.